Targeting a Pleckstrin Homology Domain with a Lysine-Reactive Covalent Binder

Area of Science:

• Biochemistry
• Medicinal Chemistry
• Pharmacology

Background:

• Bruton's Tyrosine Kinase (BTK) is a key target for treating hematological malignancies, with existing inhibitors approved by the FDA.
• Current BTK inhibitors target the ATP binding site, which is highly conserved across kinases, limiting the therapeutic index.
• The PH domain of BTK mediates its membrane recruitment and activation, presenting an alternative inhibition target.

Purpose of the Study:

• To explore a novel strategy for BTK inhibition by targeting the PH domain.
• To identify and develop inhibitors that bind to the PH domain and disrupt BTK membrane localization.
• To assess the potential for a more general approach to PH domain inhibition.

Main Methods:

• Fragment-based drug discovery was employed to identify initial binders to the BTK PH domain.
• Structure-activity relationship studies and fragment growth were conducted to optimize inhibitors.
• Crystallography was used to determine 27 crystal structures, elucidating binding modes.
• pKa values of targeted lysines in BTK and other PH domains were evaluated.

Main Results:

• A fragment was identified that covalently modifies a lysine within the inositol phosphate (PIP3) binding site of the BTK PH domain.
• This fragment was shown to inhibit the binding of a PIP3 headgroup analog to the PH domain.
• A best-in-class analog, compound 24, was developed through fragment optimization.
• Evaluation of pKa values suggests this covalent modification strategy may be applicable to other PH domains.

Conclusions:

• Targeting the PH domain offers a novel and potentially more selective approach to BTK inhibition compared to ATP-competitive inhibitors.
• Compound 24 represents a promising lead optimized through structure-based design.
• The identified mechanism of covalent modification of a lysine in the PIP3 binding site may be generalizable to inhibit other PH domain-containing proteins.